1. Field of the Invention (Technical Field)
The present invention relates to methods of making, compositions, and uses of somatostatin-derived, peptide-based radiopharmaceuticals for the diagnosis and treatment of disease, including peptide-based metal ion-labeled somatostatin-derived compositions.
2. Background Art
Peptide-Based Radiopharmaceuticals. The use of biologically active peptides, which are peptides that bind to specific cell surface receptors or that in other ways modify cellular function, has received some consideration as radiopharmaceuticals. Biospecific imaging and radiotherapy agents started with large proteins, such as antibodies, and have evolved to antibody fragments, antigen binding domain fragments and small biologically active peptides. The smaller size of biologically active peptides confers pharmacokinetic properties, such as higher target-to-non-target ratios and faster blood clearance, which are desirable for some applications.
Several peptide-based radiopharmaceutical products are in development, including those which use somatostatin-derived peptides as an imaging agent. Radiolabeled peptide analogues of somatostatin used for diagnostic imaging include .sup.123 I-labeled Tyr-3-octreotide and .sup.111 In-DTPA-octreotide imaging agents, and research is being conducted on a variety of .sup.99m Tc-labeled somatostatin analogues, including direct-labeled somatostatin analogues. An .sup.111 In-DTPA-octreotide product is commercially available in the United States and European countries, and is distributed by Mallinckrodt Medical, Inc.
Somatostatin and Analogues. Somatostatin is a hormone produced by the hypothalamus which normally inhibits the release of pituitary growth hormone. A number of peptide analogues have been developed which have pharmacological actions that mimic the naturally-occurring hormone. In normal subjects somatostatin and its analogues have the ability to suppress secretion of serotonin and the gastroenteropancreatic peptides, and growth hormone. Receptors for somatostatin are expressed on a variety of human tumors and their metastases. Somatostatin receptors have been found to be over-expressed in a wide range of tumor types including those arising in the brain (including meningioma, astrocytoma, neuroblastoma, hypophysial adenoma, paraganglioma, Merkel cell carcinoma, and gliomas), the digestive-pancreatic tract (including insulinoma, gluconoma, AUODoma, VIPoma, and colon carcinoma), lung, thyroid, mammary gland, prostate, lymph system (including both Hodgkin's and non-Hodgkin's lymphomas), and ovaries. Additionally, the tumors that most frequently produce percutaneous intrathoracic metastasis, including mammary gland tumors, lung carcinomas (especially small cell lung carcinomas), and lymphomas (Hodgkin's and non-Hodgkin's), all generally over-express somatostatin receptors which can be detected by scintigraphy (Krenning E P, Kwekkeboom D J, Bakker W H, Breeman W A, Kooij P P, et al: Somatostatin receptor scintigraphy with [111In-DTPA-D-Phe1]- and [123I-Tyr3]-octreotide; the Rotterdam experience with more than 1000 patients. Eur J Nucl Med 20: 716-731, 1993).
In spite of the high rates of over expression of somatostatin receptors on a variety of tumors, somatostatin analogues have not gained widespread clinical application for the control of cancer. Their current clinical application is primarily in the control of symptoms associated with metastatic carcinoid or VIP-secreting tumors. The somatostatin analogues have a wide therapeutic index and seem to be free of major side effects. Most of the side effects are gastrointestinal in nature and include minor nausea, bloating, diarrhea, constipation, or steatorrhea. Part of the reason for the restricted clinical use may be due to the need for long-term maintenance therapy, the consequent high cost of such therapy, and the variable effects observed in clinical settings.
Somatostatin analogues, preparation of such analogues, and uses for such analogues are known in the prior art. Such analogues are used in the treatment of certain cancers and other conditions, with one commercially available product being octreotide, manufactured by Sandoz, and sold under the trade name Sandostatin.
A wide variety of somatostatin analogues have been developed. These include RC-160, a potent somatostatin analogue originally synthesized by a team at Tulane University headed by Andrew V. Schally (Cai R Z, Szoke B, Lu E, Fu D, Redding T W and Schally A V: Synthesis and biological activity of highly potent octapeptide analogues of somatostatin. Proc Natl Acad Sci USA, 83:1896-1900, 1986). In recent studies conducted by Schally, among others, the effectiveness of RC-160 in inhibiting the growth of human glioblastomas in vitro and in vivo has been demonstrated. See, e.g., Pinski J, Schally A V, Halmos G, Szepeshazi K and Groot K: Somatostatin analogues and bombesin/gastrin-releasing peptide antagonist RC-3095 inhibit the growth of human glioblastomas in vitro and in vivo. Cancer Res 54:5895-5901, 1994.
RC-160 is a cyclic somatostatin analogue, which binds to somatostatin receptors 2 and 5 (Oberg K: Treatment of neuroendocrine tumors. Cancer Treat Rev 20:331-355, 1994). The general structure of RC-160 is as follows: ##STR1##
Other available somatostatin analogues include cyclic octapeptide analogues of somatostatin, such as ##STR2##
Peptide Radiolabeling. Peptides may be radiolabeled by a variety of means. Biologically active peptides for radiopharmaceuticals include that disclosed by Olexa S A, Knight L C and Budzynski A Z, U.S. Pat. No. 4,427,646, Use of Radiolabeled Peptide Derived From Crosslinked Fibrin to Locate Thrombi In Vivo, in which iodination is discussed as a means of radiolabeling. Peptides may be directly radioiodinated, through electrophilic substitution at reactive aromatic amino acids. lodination may also be accomplished via prelabeled reagents, in which the reagent is iodinated and purified, and then linked to the peptide. In Morgan C A Jr and Anderson D C, U.S. Pat. No. 4,986,979, Imaging Tissue Sites of Inflammation, use of chelates and direct iodination is disclosed.
The utility of DTPA and EDTA chelates covalently coupled to polypeptides and similar substances are well known in the art. Hnatowich, D J, U.S. Pat. Nos. 4,479,930 and 4,668,503. DTPA has been used as a bifunctional chelating agent for radiolabeling a variety of peptides with .sup.111 In, including .alpha.-melanocyte-simulating hormone for imaging melanoma, chemotactic peptides for infection imaging, laminin fragments for targeting tumor-associated laminin receptors and atrial natriuretic peptide for imaging atrial natriuretic receptors in the kidney.
Technetium-99m is a preferred isotope for diagnostic imaging, due to its low cost, ready availability, excellent imaging properties and high specific activities. Two approaches have been described for radiolabeling proteins and peptides with .sup.99m Tc: direct labeling and bifunctional chelates. In Dean R T, Lister-James J and Buttram S, U.S. Pat. No. 5,225,180, Technetium-99m Labeled Somatostatin-Derived Peptides for Imaging, direct labeling of somatostatin following reduction of native disulfide bonds resulting from cross-linked cysteine residues is disclosed. In U.S. Pat. No. 5,460,785, entitled Direct Labeling of Antibodies and Other Proteins with Metal Ions, referenced above, and U.S. Pat. No. 5,443,816, entitled Peptide-Metal Ion Pharmaceutical Preparation and Method, also referenced above, a variety of methods of direct labeling of peptides through sulfur-, oxygen- and nitrogen-containing amino acid sequences available for binding are disclosed.
A variety of high affinity chelates to bind .sup.99m Tc to specific sites on peptides have been developed. In one approach, the bifunctional reagent is first labeled with .sup.99m Tc, and then conjugated to the peptide. However, multiple species can result, and post-labeling purification is generally required. In another approach, a chelating agent is covalently attached to the peptide prior to radiolabeling. In Tolman G L, U.S. Pat. No. 4,732,864, Trace-Labeled Conjugates of Metallothionein and Target-Seeking Biologically Active Molecules, the use of metallothionein or metallothionein fragments conjugated to a biologically active molecule, including peptides, is disclosed. Other chelates which have been employed include a variety of N.sub.2 S.sub.2 and N.sub.3 S ligands, DTPA, and 6-hydrazinonicotinate groups.
Modes of Delivery of Radiotherapeutic Drugs. There is a need for improved methods of delivery of somatostatin-derived radiotherapeutic agents for cancer therapy because of the low absolute tumor uptake of somatostatin analogues following i.v. injection, the widespread distribution of somatostatin receptors in other tissues, and the need for highly localized therapeutic radioisotope concentrations. Some research groups have explored use of local or regional administration of radiolabeled colloid chelates and antibodies for tumor therapy (Hoefnagel C A: Anti-cancer radiopharmaceuticals. Anticancer Drugs 2:107-32, 1991). For example, in studies of brain glioblastomas, positive results have been obtained with direct intralesional radioimmunotherapy using .sup.131 I-labeled monoclonal antibodies (Riva P, Arista A, Sturiale C, Franceschi G et al: Possibility of control of malignant gliomas by direct intratumour or intralesional radioimmunotherapy (Abstract). J Nucl Med 5:144P (Abst. No. 582), 1994). With 34 evaluable patients, a median survival of 18 months was reported, versus 12 months achievable by traditional treatments, with a response rate of 38.2%, including 9 stabilized, 7 partial remission and 6 complete remission.
While use of antibodies are one treatment approach, it has become clear that another class of biologicals already possess many of the properties sought for targeting purposes. Peptide hormones and their synthetic analogues undergo high affinity interactions with target cells, and generate little or no immune response.
The targeting of somatostatin receptor-positive tumors in diagnostic imaging has a number of advantages, including the following: a) the expression of the target receptor is up-regulated in many different tumor types, and conversely the expression of receptor on normal tissues is low; b) the affinities of receptor for native hormone is high and numerous synthetic analogues which have higher affinity have been described; and c) the molecular weight of the tracer is low and circulating peptide is cleared rapidly from the circulation. The rapid clearance of the radiolabeled peptide from the circulation leads to very low backgrounds, allowing for imaging even in the face of low absolute tumor uptakes.
While it is clear that the rapid clearance of radiolabeled peptides is a considerable advantage in diagnostic imaging, it is a distinct disadvantage in targeted radiotherapy where the therapeutic effect is entirely dependent on the absolute uptake of the radionuclide at the target tumor site. Thus, intravenous administration of a radiolabeled therapeutic agent will generally not be clinically successful if the agent rapidly clears. For imaging purposes, relative uptake is important, while for therapeutic purposes, absolute uptake is important. However, local or regional administration of a radiolabeled therapeutic agent presents certain potential advantages:
a) local or regional administration sequesters and juxtaposes the peptide against the tumor, providing the highest probability of tumor binding; PA1 b) local or regional delivery may provide a physical compartment which includes the tumor, thus maximizing time the peptide is near the tumor to provide optimal irradiation of the tumor both by direct binding and non-specific local irradiation; PA1 c) local or regional delivery frequently involves regional clearance mechanisms including the lymphatic system, so that micrometastasis in regional lymph nodes can be irradiated; and PA1 d) local or regional delivery may provide rapid clearance from the blood stream, once the peptide has cleared to the blood stream, thereby minimizing irradiation to non-target organs.
Intra-Articular Use of Somatostatin for Treatment of Arthritis. In addition to those uses and potential uses for somatostatin and its analogues described above, research has indicated a potential use for it in the treatment of arthritis. In particular, the literature describes the passive, unradiolabeled, intra-articular use of somatostatin in treating rheumatoid arthritis. Fioravanti A, Franci A, Gelli R, Minari C, Montemerani M, Moscato P, and Marcolongo R: Evaluation of the efficacy of intra-articular administration of somatostatin in rheumatoid arthritis. Clin-Ter. 142(5):453-57, 1993. Another study involves the use of gold salts and somatostatin to form a new combined treatment for psoriatic arthritis. Matucci-Cerinic M, Pignone A, Lotti T, Partsch G, Livi R, and Cagnoni M: Gold salts and somatostatin: a new combined analgesic treatment for psoriatic arthritis. Drugs-Exptl.-Clin.-Res., 18(2):53-61 (1992). The literature also describes radiation synovectomy using radiocolloids. See, e.g., Chinol M, Vallabhajosula S, Goldsmith S J, Klein M J, Deutsch K F, Chinen L K, Broadack J W, Deutsch E A, Watson B A, and Tofe A J: Chemistry and biological behavior of samarium-153 and rhenium-186-labeled hydroxyapatite particles: potential radiopharmaceuticals for radiation synovectomy. J. Nucl. Med., 34:1536-1542 (1993). See also, Deutsch E, Brodack J W, Deutsch K F: Radiation synovectomy revisited. Eur. J Nucl. Med., 45:1113-1127 (1993). Radiation synovectomy consists of the intra-articular injection of a beta-emitting radiopharmaceutical to counteract and control synovial inflammation. The use of radiocolloids has been predicated on the direct juxtapositioning of the radioactive material against the synovial membranes in joints, and by an active process of colloid uptake by the cells of the synovial membrane. In some applications, colloids are preferred over more soluble forms such as particulates, because the use of colloids helps to restrict radioactivity to the joint without leakage. Such leakage can lead to high accumulations in the regional lymph nodes, and to a lesser extent the lungs, and thereby result in unacceptable radiation to non-target organs. Use of a soluble form may therefore cause excessive, unwanted whole-body radiation. Similarly, administration via the blood may not target the appropriate cells and also lead to high non-target uptake. The concerns of practitioners have been that this treatment is expected to be a repeated treatment, and will therefore necessitate administration of radioactivity to other tissues. Some of the advantages of using .sup.188 Re for radiation synovectomy have been described in Wang S J, Lin W Y, Hsieh B T, Shen L H, Tsai Z T, Ting G, and Knapp F F, Jr.: Rhenium-188 sulphur colloid as a radiation synovectomy agent. Eur. J. Nuc. Med. 22:505-507 (1995).
The Use of Ascorbate and Similar "Stabilizers" for Radiopharmaceuticals. Radiopharmaceutical compositions are known to degrade after radiolabeling by oxidation and by autoradiolysis. Some radiopharmaceuticals, such as technetium-99m and rhenium-186 or rhenium-188 labeled compounds, are known to require stabilizing agents such as antioxidants or reducing agents to maintain the radionuclide in a suitable oxidation state. Both technetium and rhenium normally exist in their highest or +7 oxidation state, which is their stable state, until they are reduced with stannous or other reducing agents in radiopharmaceutical kits. The labeled or complexed radiopharmaceutical kit becomes unstable if the complexed reduced isotope is oxidized to a higher oxidation state, releasing the bound isotope from the ligand as free (unbound) pertechnetate +7 or free perrhenate +7. Compounds such as ascorbic acid, gentisic acid, and others have been used to inhibit the oxidation of the radionuclide and/or reducing agent. In particular, the use of antioxidants, typically ascorbic and gentisic acid, is described in the literature for the purpose of extending shelf lives of low reduction-capacity, stannous-containing, radiopharmaceutical kits.
As used herein, the term "autoradiolysis" includes chemical decomposition of a peptide or protein by the action of radiation emitted from the radioisotope coupled to the peptide or protein. Autoradiolysis may be caused by the formation of free radicals in the water or other medium by the radiation emitted from the radionuclide. Free radicals are molecules or atoms containing a single unpaired electron, and exhibit high chemical reactivity. The action of antioxidants as radiopharmaceutical kit stabilizing agents involves their action as "free radical scavengers", as is generally known in the art. Ascorbic acid and gentisic acid act as free radical scavengers by donating reactive hydrogen atoms to the free radical intermediates yielding a non-reactive molecule (Kowalsky, R. J. and Perry, J. R, Radiopharmaceuticals in Nuclear Medicine Practice, Connecticut: Appleton and Lange 1987, 88-89). Autoradiolysis can be a significant problem with rhenium isotopes, and is typically somewhat less of a problem with technetium.
The traditional techniques of adding HSA to a composition or keeping it frozen between preparation and use are not always effective or practical for use with many radiolabeled peptides and proteins. Despite the promise shown by a number of newly-developed peptides for diagnostic and therapeutic applications, their susceptibility to autoradiolysis may limit their use. Therefore, the development of effective but non-damaging stabilizing agents is a significant and much-needed advancement in the art.